Programmed cell death plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms. The susceptibility to apoptosis is regulated by intracellular checkpoints of which the Bcl-2 family plays a prominent role. Recently, the Bcl-2 family has markedly expanded and possess both positive and negative regulators of cell death. The ration of death antagonists (Bcl-2, Bcl-xL, Mcl-1 A1) to antagonists (Bax, Bak, Bcl-xz, Bad) dictates the response to an apoptotic signal. Family members function, at least in part, through protein-protein interactions. For example, BAX heterodimerizes with BCL-2 and also forms homodimers; BAX counters BCL-2's protection and when in excess induces apoptosis. Evolutionarily conserved domains BH1-4 are found in various members and corresponds to (alpha) helices 1-7. Intact BH1 and BH2 are critical for death antagonists (BCL-2) to dimerize with agonists (BAX) and to repress death; whereas, BH3 is critical for death agonists (BAX) to promote death and dimerize with antagonists (BCL-2). To date, most of the progress in understanding cell death has been horizontal, an expanded set of proteins at each step. However, protein interactive cloning has begun to vertically integrate this pathway. Using BCL-2 as bait, BAD was identified and selectively dimerizes with BCL-xL or BCL-2 displacing BAX and restoring apoptosis. BAD is phosphorylated following IL-3 and itself binds 14-3-3 establishing a link between survival factors and the BCL-2 checkpoint. BAX which appears to lie downstream has a partner BID which only possesses the BH3 "Death domain " and promotes apoptosis. The future goal is to link the BCL-2 checkpoint to proximal signal transduction and distal death effector mechanism. Toward that end: 1) The functional significance of BAD phosphorylation and its interactions with 14-3-3 versus Bcl-xL will be characterized. 2) The functional role of BID within apoptosis will be delineated, and 3) A minimal death domain will be defined and the mechanism of killing by each BCL-2 family death agonist will be detailed.